CS257289B2 - Preparation method of complex compound of at least one flavanolignane with at least one phospholipide - Google Patents

Abstract

The invention relates to novel compounds comprising lipophilic complexes of silybin, silidianin, and silicristin with phospholipids, and the preparation of these complexes by non-conventional methods. Absorption of the novel compounds in the gastrointestinal tract is appreciably greater, resulting in higher plasma levels than for the individual flavanolignanes. The resulting improvement in the pharmacokinetic and pharmacological parameters is such that the substances can advantageously be used in the treatment of acute and chronic liver disease of toxic, metabolic or infective origin or of degenerative nature.

Description

The invention relates to a process for the preparation of novel phospholipid complexes with the main components of silymarin, a known standardized extract obtainable from the seeds of Silybum marianum. The complexes are useful in the treatment of liver diseases of various origins. Silymarin contains three main components: silybin, silidianin and silicristin, which are believed to be the active ingredient of the extract with respect to the therapeutic and protective action on the liver. The main component is silybin, which is a mixture of two diastereomers (Chem. Commun. 696, 1979) in a weight ratio of approximately 1: 1, for which there is a large amount of literature on pharmacotoxicology and clinical use with regard to water-soluble hemiesuccinic diester .

The efficacy of silybin and other components of silymarin, especially when injected, occurs in liver cells where flavanolignans are involved in the process of stabilizing and protecting the liver cell membrane against adverse agents such as carbon tetrachloride, falloidin, amanitin, certain heavy metals, galactosamine and such as various antibiotics that release enzymes in their action, causing necrosis of the liver. Some of these harmful substances simulate hepatic damage caused by viruses that cause common forms of hapatitis in humans, suggesting the therapeutic importance of antihepatotoxic molecules of the species.

It is known in the literature that the best protection against the above-mentioned adverse agents is intraperitoneal or intravenous administration of the compounds as fast as possible to achieve high concentrations in the bloodstream and in the protected organ. Despite occasional reports and oral absorption of silybin (Arzneim. Forsch. 23, 1322, 1973; Arzneim. Forsch. 25, 902, 1975; Planta Medica 45, 216, 1982), relevant pharmacological investigations are inadequate and difficult to reproduce. This demonstrates that the active substance does not have a sufficiently high or adequate biological activity when administered by this method.

The invention relates to a process for the preparation of novel compounds by the chemical reaction of at least one phospholipid and at least one of the above flavanolignan.

The compounds prepared by the process of the invention have the general formula I

_Of CH ² -0-R ^ CH-OR

/AND/

where R and R1 may have the same or different meanings and represent an acyl radical of palmitic, stearic, oleic, linoleic or linolenic acid, R2 represents a group of the formula (R ₎ . Ф <£ 1

-CH ₂ -CH ₂ -N (CH ₃ ) ₃ -CH ₂ CH ₂ -NH ₃ -CH ₂ CH (COOH) -NH ₃ and R ₃ represents a group of formula a, b or c

(a) (b)

wherein Me is methyl, wherein the weight ratio between flavanolignans and phospholipids can vary from 1: 0.3 to 1: 2.

The phospholipids used in the compositions may be of natural or synthetic origin. Preferably, natural phospholipids of plant origin (soybean phospholipids) are used, such as (R) is, for example, Lipoid S 10 ' ⁷ (Lipoid from KG-Ludwigshafen, Germany) having at least 90% phosphatidylcholine containing an average of 63% linoleic acid, 16% palmitic acid, 3.5% stearic acid and 11% oleic acid, based on the total fatty acid content; natural phospholipids extracted from the liver may also be used.

The complex compounds prepared by the process of the invention have a pronounced lipophilic character and, surprisingly, unexpectedly improve the oral absorption of complex flavanolignan and thus exhibit improved specific efficacy in various pharmacological tests performed by experimental methods already described in the literature on the occasion of testing the parent compounds.

According to the process of the invention, the novel complex compounds are prepared by reacting 0.3 mol to 2 mol, preferably but 1 mol, of natural or synthetic phospholipids, which may be, for example, phosphatidylcholine, phosphatidylethanolamine or phosphatidylserine, with one mol of silybin, silidianin or silicristin. , either singly or in natural mixtures (silymarin), in aprotic organic solvents such as dioxane or acetone; from the solvent, the complex prepared compound can be isolated by precipitation with a non-solvent such as aliphatic hydrocarbons or by freeze drying or spray drying.

Known methods for preparing liposomal active complexes are based on the presence of water or a buffer solution. In contrast, the process according to the invention uses only a solvent having a reduced dielectric constant. While the starting molecules (silybin, silidianin, etc.) are insoluble in chloroform, ethyl ether or benzene, they are extremely soluble in these solvents upon complexation with the phospholipid. This change in chemical and physical properties is due to the creation of a true permanent complex

13 of the compound as shown by the changes in the IR, 1 H-NMR and C-NMR spectra of the compounds after the complex of the invention was formed.

The spectroscopic characteristics of the complex compound are distinctly different from those of the individual components and demonstrate a profound interaction between the flavanolignans and the polar ends of the phospholipids. For example, the IR spectrum of phosphatidylcholine (particularly dipalmitoyl phosphatidylcholine) at 1250 cm ^- ^ shows a band due to the group P = O; this band disappears in the 1: 1 spectrum of the silybin complex while in the mechanical mixture of the two components.

The formation of the complex compound is also illustrated by the NMR spectrum and relaxation times of the hydrogen and carbon atoms involved in the formation of bonds between the complexing compounds. In particular, the proton spectrum shows a substantial spread of the signals of hydroxyl protons, aromatic protons and methoxy groups of flavanolignans and the NMe-lipid group. On the other hand, the 1 H-NMR spectrum of a mixture of phosphatidylcholine and silybin without forming a complex compound is a simple sum of the spectra of both components.

In the ¹³ C spectrum, the relaxation time of the core most closely involved in the formation of the complex compound is sufficiently reduced to eliminate all signals of the flavanolignan and choline group and glycerine group of the phospholipid.

It is therefore believed, without being bound by the scope of the invention, that silybin binds to the phospholipid via its polar end so tightly as to inhibit inert or intramolecular rotation. On the other hand, the non-polar portion of the lipid that is not involved in bonding is freely movable, so that the complex compound is highly liposolubile.

Silidianin and silicristin have physical and chemical properties similar to gilybin; the same is true for silymarin, which has three active centers mixed with each other. Moreover, the above physical and chemical considerations are also applicable, with certain quantitative differences, to flavalignan phospholipid complex compounds with a molar ratio different from 1: 1.

As mentioned above, from the biological point of view, these flavanolignan phospholipid complex compounds are unexpectedly biologically more suitable for oral administration, thereby avoiding the known absorption problems commonly encountered with many phenolic compounds and especially silymarin. Therefore, the pharmacological efficacy of the novel compounds prepared by the process of the invention is much more obvious and demonstrable even when administered orally.

As shown in Table I (concerning silybin and its 1: 1 complex compound with phosphatidylcholine from soy phospholipid), absorption in rats is significantly more favorable in the complex form. Other components of silymarin behave similarly to silybin when administered in a complex form.

Table II demonstrates the efficacy of the same 1: 1 complex compound of silybin with phosphatidylcholine from soy phospholipid in a conventional liver carbonate poisoning test. only a slight decrease in these enzymes. Similar results are shown in Table III concerning the 1: 1 complex compound of silymarin with phosphatidylcholine from soy phospholipids.

Also of great importance is the pharmacokinetic comparison in humans after oral administration of equimolar doses of silybin and its 1: 1 complex compound with phosphatidylcholine from soy phospholipids; see Table IV.

Notes to Table I ⁺ equivalent to 200 mg / kg silybin

0.00 not determinable as it is below 0.05 µg / ml

Notes to Table IV

0.00 below the sensitivity of the method (35 ng / ml)

Table I

Pharmacokinetic Comparison of Equimolar Dose of Silybin and 1: 1 Silybin Phosphatidylcholine Complex in Oral Rats

Sampling time (hours)

Aig / ml

Table I continued

Treatment Dose Number mg / kg animals

pers 0.25 h 0.50 h Complex 520 « 4 4.12 + 3.69 + 1.59 .0,55 silybin 200 4 0.07 + 0.06 + 0.01 0.01

1.00 h 2.00 h 3.00 h 4.00 h 6.00 h 8.00 h 4.60 Ί- 2.00 0.38 + 0.11 + 0.05 + 0.00 + 87, 87 0.09 0.09 0.02 0,00 0,00 0.11 + 0.05 + 0.05 + 0,00 + 0,00 + 0.2 0.00 0.00 - -

Table II

Antihepatotoxic efficacy of silybin and 1: 1 silybin phosphatidylcholine complex in carbon tetrachloride poisoning rats

Treatment Dose mg / kg p Number of animals per group GPT HIVE GOTH HIVE Normal control - 10 22.40 ° ’+ 1.93 88.50 ° * · 5.20 poisoned control - 10 145.40 + 31.43 339.40 + 59.07 Silybin 25 10 94.30 + 21.87 (-35.2%) 252.60 + 48.04 (-25.6%) Silybin 250 10 94.90 + 31.64 (-34.8%) 200.00 ’+ 33.01 (-41.1%) complex 65,8 * 10 65.90 + 20.49 (-54.7%) 181.80 + 52.65 (-46.4%) complex 658 ** 10 46.90 °° + 9.61 (-67.8%) 148.10 * * - 16.96 (-56.8%)

P <0.05x equivalent for 25 mg silybin

P <0.01x equivalent for 250 mg silybin

Table III

Antihepatotoxic efficacy of silymarin and 1: 1 silymarin phosphatidylcholine complex in carbon tetrachloride poisoning rats

Treatment Dose mg / kg p Number of animals per group GPT HIVE GOT U / L Normal control - 10 26.33 ** + 2.26 105.44 ** + 4.98 poisoned controls - 10 146.50 + 45.10 358.50 + 64.15 Silymarin 25 10 125.30 + 32.20 (-1475) 290.15 + 38.52 (-1971) Silymarin 250 10 82.44 + 21.90 (-43 7 7) 190.70 * + 28.85 (-46.8) Complex 65 ^a 10 110.22 + 18.75 (-24.8) 247.50 + 26.52 (-3170) Complex 650 ^p 10 50.48 * + 8.52 (-55.0) 201.38 * + 23.64 (-43.8)

x P <0.05 XX P <0.01

(a) equivalent for 25 mg silymarin

(b) equivalent to 250 mg silymarin

Table IV

Pharmacokinetic comparison of equimolar doses of silybin and 1: 1 silybinphosphatidylcholine complex in humans

Treatment Dose per os Number of patients 0.25 h Sampling time / hours / nq / ml 6,00 h 8,00 h 0.50 h 1.00 h 2.00 h 3.00 h 4.00 h Complex 700 4 0.00 46 79 79 108 112 50 40 Silybin 270 4 ' 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00

The compound prepared by the method of the invention is suitable for the treatment of acute or chronic liver diseases of toxic, metabolic and / or infectious origin, degenerative liver diseases and preventive treatments against liver damage caused by the use of drugs and / or other substances adversely affecting the liver. The compounds prepared by the process of the invention may be used in conventional oral pharmaceutical formulations such as pills, dragees, capsules or packs and in the form of suspensions.

The proposed daily dose for adults of a 1: 1 complex compound of silybin and soy phosphatidylcholine is 130 to 1300 mg, corresponding to 50 to 500 mg of silybin.

The following examples illustrate the invention but do not limit it in any way.

Example 1

1: 1 silylbeta / dipalmitoylphosphatidylcholine complex compound (I, with R = palmitoyl;

R 1p = -CH ₂ -CH ₂ -N (CH ₃ ) ₃ ; R ₃ = (a) J

4.82 g (0.010 mol) of silybin are dissolved in 150 ml of boiling acetone and treated with 8.06 g (0.011 mol) of dipalmitoylphosphatidylcholine. The resulting solution was refluxed for one hour and then concentrated in vacuo to a volume of 30 mL. The concentrated solution was poured into 350 ml of n-hexane with stirring, allowed to stand overnight at room temperature, then the solid precipitate was collected by filtration, washed with n-hexane and dried in vacuo at 40 ° C. The product was 11.6 g of the complex compound (95% yield) as a yellowish-white powder.

E _1% = 175.8 at 288 nm (CH ₃₀ OH)

Analysis for ^C 65 ^H 10 ^NO 18 ^P (MW 1,216.48) calculated% C 64.18 H 8.45 N1.15 P 2.55 found% C 63.97 H 8.47 N 1.11 P 2 , 51

Example 2

1: 1 silicristidine / dipalmitoylphosphatidyeltanolamine complex compound (I, with R = R ^ = = palmitoyl; R ® = -CH ₂ -CH ₂ - ^ H ₃ ; R ₃ = (b))

A suspension of 4.82 g (0.010 mol) of silicristidine in 100 ml dioxane is treated at room temperature with stirring with a solution containing 6.91 g (0.010 mol) dipalmitoylphosphatidylethanolamine in 200 ml dioxane. The mixture was allowed to react for 5 hours and freeze-dried. The product was 11.7 g of the complex compound as a yellowish-white powder. ,

Δ _χ% = 174.6 pfci 288 nm (CH-jOH). ,

Analysis calculated for ^C 52 ^H 96 ^NO 18 ^P (MW 1,174.40)% С 63.41 Η 8.24 N1.19

Ρ 2.63 found% С 63.27 Η 8.26 Ν 1.17 Ρ 2.59

Example 3

1: 1 silydianin / distearoylphosphatidylcholine complex compound (I, with R = =

- stearyl; R 8 = -CH ₂ -CH ₂ - $ (CH ₃ ) ₃ ; R ₃ = (c)

A suspension of 4.82 g (0.010 mol) of silidianin in 150 ml of acetone was refluxed with stirring with 8.68 g (0.011 mol) of distearoylphosphatidylcholine for approximately one hour. The reaction mixture was concentrated in vacuo to a volume of 35 mL and diluted with 400 mL of n-hexane. After standing overnight at room temperature, the solid precipitate was collected by filtration, washed with n-hexane and dried under vacuum at 40 ° C. The product is 11.1 g of a yellow-white complex compound (yield 87.4%).

^E 1% ^{= 148/8} At ^{288 nm} (ch ₃ oh)

Analysis calculated for% C, 65.12; H, 8.71; N, 1.10; 2.43 found:% C, 64.98; H, 8.73; N, 1.12, 2.48.

Example 4

1: silybin / phosphatidylcholine complex compound (soybean phosphatidylcholine)

A suspension of 4.82 g of silybin (0.010 mol) in 150 ml of acetone is treated at room temperature with stirring 9.2 g (0.012 mol) of Lipoid S 100 (MW 770). The reaction mixture became clear in three hours and concentrated in vacuo to a volume of 30 ml. After dilution with 300 ml of n-hexane, the complex compound precipitates and is collected by filtration after standing overnight and dried under vacuum at 40 ° C. Yield: 11.9 g (94% of theory) of the product as a yellowish white powder. ' ^E 1% ^{= 172/8} At ^{288 nm} (CH3OH)

Analysis (MW 1,252) calculated% N 1.12 P 2.48 found% N 1.15 P 2.55

Example5

1: 1 silymarin / phosphatidylcholine complex compound (soybean phosphatidylcholine)

A solution of 5 g silymarin in 100 ml acetone is treated with stirring at room temperature (R) g Lipoid S 100. After complete dissolution, the reaction mixture was concentrated in vacuo to a volume of ml and poured into 300 ml of ligroin with stirring; The precipitate which was allowed to stand overnight was collected by filtration, washed with ligroin and dried under vacuum at 40 ° C. The product is 11.1 g of the complex compound.

^E 1% ^{= 170/2} At ^{288 nm} (ch ₃ ° h)

Analysis: Found% N 1.12 P 2.50

Example 6

1: 2 silybin / phosphatidylcholine complex compound (soybean phosphatidylcholine)

A suspension of 4.82 g of silybin (0.010 mol) in 75 ml of dioxane is treated with stirring with a solution of R (R) containing 15.4 g (0.020 mol) of Lipoid S 100. The reaction mixture became clear in four hours and freeze-dried to give 20 g of a pale yellow complex compound.

E _1% = 160 at 288 nm (CH-OH)

Analysis (molecular weight 2,022)

Calculated% N 1.38 P 3.07 found% N 1.35 P 3.11

Example 7 "

11,3 silybin / phosphatidylcholine complex compound (soybean phosphatidylcholine)

A solution containing 2.41 g (0.005 mol) of silybin and 100 ml of dioxane is treated at 60 ° C with 0.770 g (0.001 mol) of Lipoid S 100 for one hour. The reaction mixture is evaporated to dryness in vacuo and the residue is dissolved in 100 ml of chloroform; unreacted silybin to the complex compound is collected by filtration and the mother liquor containing the complex compound is evaporated to dryness in vacuo. The residue, dried under vacuum at 30 ° C, represented 2.3 complex compounds as a yellowish-white powder.

E _1% = 300,288 nm (CH ₃ OH) ₇

Analysis (Molecular Weight 713) calculated% N 0.59 P 1.30 found% N 0.56 P 1.34

Example '

Pill containing silybin / phosphatidylcholine complex compound (soybean phosphatidylcholine)

Each 1.2 g pill contains:

complex compound (corresponding to 200 mg silybin) granulated cellulose lactose starch • PVP carboxymethyl starch magnesium stearate

520 mg

90 mg

100 mg

100 mg mg mg mg

Example 9.

Capsules containing 1: 1 silybin / phosphatidylcholine complex compound (soybean phosphatidylcholine)

Each capsule contains at its own weight 0.275 g a complex compound (corresponding to 100 mg silybin) powdered silica

Magnesium stearate

260 mg mg

2.5 mg

2.5 mg

Example 10

Water Suspension Granulate containing 1: 1 silybin / phosphatidylcholine complex compound (soybean phostatidylcholine)

Each 3 g pack contains:

complex (corresponding to 200 mg silybin) 520 mg lactose 2 000 mg mannitol 238 mg ammonium glycyrrhizinate 10 mg natriumsacharin 2 mg dried orange juice 200 mg flavoring substance 30 mg

Example 11

Capsules containing 1: 1 silymarin / phosphatidylcholine complex compound (phosphatides1choline from soybean)

Each capsule is the same as in Example 3 but contains 260 mg of a 1: 1 silymarin / phosphatidylcholine complex compound (corresponding to 100 mg silymarin) instead of the silybin complex compound shown in Example 9.

Example 12

Granules for suspending in water containing 1: 1 siLymarin / phosphatidylcholine complex with Loučín

Each 3 g package has the same composition as the package of Example 10 but contains 520 mg of a 1: 1 silymarin / phosphatidylcholine complex compound (corresponding to 200 mg silymarin) instead of the silybin complex compound shown in Example 10.

Claims (2)

OBJECT OF THE INVENTION A process for the preparation of a phospholipid complex compound of formula I of at least one flavanolignan with at least one CH ₃ -OR ¹² CH-OR ₁ .(TO _х - ^й 3 VYr r Y X) H OH 0 Where I means R and R ^ are the same or different acyl groups stearate, oil, linoleic or linolene, selected from the group consisting of palmite, one of the groups of the formula -CH ₂ -CH ₂ - $ (CH ₃ ) ₃ , -CH ₂ CH ₂ -NNE ₃ -CH ₂ CH (COOH) R _{3 is} a, b or c Where Me is methyl, at a molar ratio between flavanolignans and phospholipids of 1: 0.3 to 1: 2, characterized in that at least one natural or synthetic phospholipid of the formula II is reacted CH _o -OR I ² ch-or ₂ CH - OP ² · o (II) wherein R 1 and R ₂ are as defined above, in an aprotic solvent with at least one flavanolignan of the general formula III / III (wherein R 2 is as defined above), in the molar ratio of flavanolignan to phospholipid 1: 0.3 to 1: 2 and the resulting complex compound is isolated by non-solvent precipitation, freeze-drying or spray-drying. 2. Process according to claim 1, characterized in that dioxin or acetone is used as the aprotic solvent and aliphatic hydrocarbons as non-solvents individually or in the form of mixtures.